1) The evolution of nervous system patterning: insights from sea urchin development. Lynne Angerer (15%) in collaboration with Robert D. Burke, Shunsuke Yaguchi and Robert Angerer. We published this review in Development 138:3613-23. It discusses recently elucidated mechanisms that localize and pattern the nervous system of sea urchin embryos. These include the recent findings that there are two overlapping regions of neurogenic potential at the beginning of embryogenesi that are remodeled by separate, yet linked, signals, including Wnt and subsequently Nodal and BMP. These signals act to specify and localize the anterior and ciliary band neural fields. Comparison of the evolution of these patterning signals highlights the extreme conservation of mechanisms underlying the processes that set up neuroectoderm territories in deuterostomes. 2) Control of Wnt signaling in the anterior neuroectoderm. (15%) (Ryan Range and Lynne Angerer) Our objective was to determine how Wnt signaling controls the development of regions that will give rise to neurons (neuroectoderm) versus those that do not. Neuroectoderm forms where Wnt is antagonized and epidermal ectoderm differentiates where Wnt is active. At least three different Wnt pathways, Wnt/&#946;-catenin, Wnt/PCP and Wnt/Ca+2, set up these two types of ectoderm and at least three different regulators of Wnt signaling, Dkk1, sFRP1/5 and Dkk3 are expressed in the anterior neuroectoderm where Wnt signaling is low. Two different Wnt receptors, Frizzled 5/8 and Frizzled 1/2/7, function in at least two of these pathways and they mediate antagonistic activities with respect to ectoderm patterning. Frizzled 5/8 activity converts neuroectoderm except that at the anterior end to epidermis while Frizzled 1/2/7 activity slows down this Frizzled 5/8-dependent function, allowing accumulation of neuroectoderm regulatory proteins. When Frizzled1/2/7 expression is down regulated at the anterior end of the embryo, another Frizzled5/8 inhibitor, Dkk1 is up regulated protecting conversion of neuroectoderm to non-neural ectoderm fates. These studies have revealed a set of unexpected and surprisingly complex interactions among different Wnt pathways that function in early patterning. Manuscript in preparation. 3) Role of individual Wnt ligands in ectoderm patterning. (10%) (Zheng Wei, Ryan Range and Lynne Angerer) Surprisingly we found that Wnt1 activity is required at a relatively late stage to maintain the correct orientation of oral and aboral tissues that form on the ventral and dorsal sides of the embryo. In embryos lacking Wnt1, nodal is ectopically expressed in posterior ventral ectoderm and endoderm and its signaling converts their fates to oral ectoderm. As a consequence the ciliary band position shifts from the ventral to the dorsal side of the blastopore or anus. And the blastopore, which marks the posterior pole of the embryo, is now at the posterior edge of the oral ectoderm near the mouth, an anterior structure, as a result of the exaggerated morphological distortions of the embryo. This work shows that continued interactions between Wnt and Nodal signaling are required to maintain the body plan of the embryo. Manuscript, submitted. 4) De novo neurogenesis in the foregut. (10%) (Zheng Wei, Lynne Angerer) We made the surprising discovery that pharyngeal neurons of sea urchin embryos develop de novo from foregut endoderm through the activity of the transcription factors, Six3 and Nkx3-2. We ruled out migration of ectodermal cells to the pharynx by tracking all presumptive ectoderm cells with the photo-activatable protein, KikGR. Neurons appear in the foregut even when it does not join with ectoderm to form the mouth. We showed that Six3 is expressed transiently in foregut precursors and established that Six3 is required for Nkx3-2, which is required for foregut neural differentiation. These findings reveal that botth endodermal and neural gene regulatory networks operate in foregut cell lineages. This pluripotency may be facilitated by the activity of SoxB1, an ortholog of vertebrate factors shown to maintain a neural precursor state. These results challenge a fundamental concept in developmental biology that nerves develop only from ectoderm. This work was published this year in Proc. Nat. Acad. Sci USA, 108, 9143-9147. 5) De novo neurogenesis in the foregut in sea star embryos. (5%) (Rocio Benabentos and Lynne Angerer) This project was initiated in June, 2011, to determine if de novo pharyngeal neurogenesis also operates in an echinoderm that diverged approximately 500 mya. Neurons are present in the pharynx and experiments tracking their origins and testing whether the same gene regulatory pathway operates in these cells are underway. 6) Mechanisms underlying endomesoderm segregation. (15%) (Adi Sethi, Lynne Angerer) Although, in vertebrate embryos, Wnt/beta-catenin signaling is known to be required for endomesoderm specification and Notch is implicated in controlling the balance between endoderm and mesoderm, how these actually work in the transition from endomesoderm progenitor to stably committed endoderm and mesoderm is not understood. In sea urchin embryos, endomesoderm segregation requires sequential Notch and Wnt/beta-catenin signaling, which restricts endoderm and mesoderm gene regulatory networks operating simultaneously in endomesoderm progenitors, specifically to endoderm and mesoderm lineages. Notch initiates segregation in mesoderm progenitors by inhibiting expression of the transcription factor, Hox11/13b, which drives a key early endoderm regulatory circuit. This circuit subsequently activates transcription of the Wnt/beta-catenin ligand, Wnt1, only in the presumptive endoderm because Notch inactivates the circuit in the mesoderm. The Wnt1-dependent Wnt/beta-catenin circuit reinforces the distinction between endoderm and mesoderm. Finally, Notch signals promote the nuclear export of the beta-catenin binding partner, TCF, thereby completely insulating the mesoderm from Wnt/beta-catenin activity and an endoderm fate. This three-step mechanism generates optimal signaling environments in endoderm and mesoderm and may constitute a mechanism used in many embryos to achieve endomesoderm segregation. Manuscript, in review. 7) Dopaminergic neurons regulate the embryos response to food density (15%) (Diane Adams, Lynne Angerer) Previous work with pharmacological inhibitors of dopamine receptor function suggested that dopamine signaling was involved in the embryos response to food density. We have confirmed this hypothesis by perturbing this pathway at the level of dopamine production, dopamine activity and by eliminating a dopamine D2 receptor. The surprising finding from this work is that the default developmental program, which occurs in the absence of food, supports the growth of long arms. In contrast, when dopamine signaling is stimulated, which occurs at high food densities, the developmental program is suppressed. Thus, the commonly held view that the developmental plasticity involves growth of longer larval arms to optimize food gathering potential is incorrect;instead plasticity relies on a signaling pathway that works in the opposite direction, to inhibit arm growth. Thus, selection for developmental plasticity is not to enhance food gathering potential, but to favor conservation of maternal reserves. Consistent with this hypothesis, we found that embryos with long arms have a significant loss of lipid reserves. Because neurons producing dopamine are positioned near the points of skeletal growth, they are excellent candidates for mediating the skeletal growth response. A manuscript reporting this novel mechanism of developmental plasticity is in final revision in Nature Communications.